Opioids are an effective treatment for chronic pain, but repeated use can result in tolerance and physical dependence. These changes are thought to require activation of NMDA receptors and nitric oxide synthase (NOS), because blockade of either can prevent induction of tolerance and dependence. Both of these processes are blocked by the small molecule agmatine (decarboxylated arginine), an endogenous NMDA-receptor antagonist and NOS inhibitor. Because the NMDA-R/NOS system is considered a likely mediator of agmatine's protective effect, the proposed study will explicitly compare its action with that of NMDA-R antagonists and NOS inhibitors as well as searching for other target proteins or proteins altered as a consequence of its action. The proposed project will apply mass spectral phosphoproteomic analysis to compare the complement of proteins phosphorylated in morphine tolerance with those phosphorylated when agmatine or other NMDA-R/NOS inhibitors protects subjects from tolerance. The project will test the hypothesis that agmatine blocks the development of spinal morphine tolerance through signal transduction pathways distinct from the NMDA-R/NOS cascade. The first aim will compare agmatine effects with those of other NMDA-R/NOS inhibitors. The second aim will add co-administered morphine to the first aim and identify the phosphorylation cascades specific to morphine tolerance using 2D LC- MS/MS on spinal cord extract. The third aim will validate these changes and determine the neuronal or glial localization of the altered phosphopeptides. The results of these studies should significantly extend our mechanistic understanding of agmatine modulation of neuroplasticity in opioid tolerance. Future studies could then focus on targeting the identified phosphorylation cascades in a broad spectrum of CNS maladaptive phenomena including opioid self-administration and tolerance, chronic pain, and spinal cord injury. The Public Health Relevance: Tolerance to opioid analgesics remains a key factor limiting access of chronic pain sufferers to adequate relief. This project will apply phosphoprotein analysis to identify the signaling processes activated or suppressed as chronic morphine tolerance develops in rodent spinal cord. Compounds that block spinal tolerance and synaptic plasticity by antagonizing NMDA receptors or inhibiting nitric oxide synthase will be the primary focus.